Methyl-CpG binding domain (MBD)2/3 specifically recognizes and binds to the genomic mCpG site with a β-sheet in the MBD to affect embryonic development in Bombyx mori

Methyl-CpG (mCpG) binding domain (MBD) proteins especially bind with methylated DNA, and are involved in many important biological processes; however, the binding mechanism between insect MBD2/3 and mCpG remains unclear. In this study, we identified 2 isoforms of the MBD2/3 gene in Bombyx mori, MBD2/3-S and MBD2/3-L. Binding analysis of MBD2/3-L, MBD2/3-S, and 7 mutant MBD2/3-L proteins deficient in beta 1-beta 6 or a1 in the MBD showed that beta 2-beta 3-turns in the beta-sheet of the MBD are necessary for the formation of the MBD2/3-mCpG complex; furthermore, other secondary structures, namely, beta 4-beta 6 and an a-helix, play a role in stabilizing the beta-sheet structure to ensure that the MBD is able to bind mCpG. In addition, sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes. Furthermore, MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B. mori embryonic development, similar to that of DNA methylation; however, MBD2/3-S without beta 4-beta 6 and a-helix does not alter embryonic development. These results suggest that MBD2/3L recognizes and binds to mCpG through the intact beta-sheet structure in its MBD, thus ensuring silkworm embryonic development.